The present invention relates to the identification and characterisation of insect steroid receptors from the Lepidoptera species Heliothis virescens, and the nucleic acid encoding therefor. The present invention also relates to the use of such receptors, and such nucleic acid, particularly, but not exclusively, in screening methods, and gene switches. By gene switch we mean a gene sequence which is responsive to an applied exogenous chemical inducer enabling external control of expression of the gene controlled by said gene sequence.
Lipophilic hormones such as steroids induce changes in gene expression to elicit profound effects on growth, cellular differentiation, and homeostasis. These hormones recognise intracellular receptors that share a common modular structure consisting of three main functional domains: a variable amino terminal region that contains a transactivation domain, a DNA binding domain, and a ligand binding domain on the carboxyl side of the molecule. The DNA binding domain contains nine invariant cysteines, eight of which are involved in zinc coordination to form a two-finger structure. In the nucleus the hormone-receptor complex binds to specific enhancer-like sequences called hormone response elements (HREs) to modulate transcription of target genes.
The field of insect steroid research has undergone a revolution in the last three years as a result of the cloning and preliminary characterisation of the first steroid receptor member genes. These developments suggest the time is ripe to try to use this knowledge to improve our tools in the constant fight against insect pests. Most of the research carried out on the molecular biology of the steroid receptor superfamily has been on Drosophila melanogaster (Diptera), see for example International Patent Publication No WO91/13167, with some in Manduca and Galleria (Lepidoptera).
It has been three decades since 20-hydroxyecdysone was first isolated and shown to be involved in the regulation of development of insects. Since then work has been carried out to try to understand the pathway by which this small hydrophobic molecule regulates a number of activities. By the early 1970s, through the studies of Clever and Ashburner, it was clear that at least in the salivary glands of third instar Drosophila larvae, the application of ecdysone lead to the reproducible activation of over a hundred genes. The ecdysone receptor in this pathway is involved in the regulation of two classes of genes: a small class (early genes) which are induced by the ecdysone receptor and a large class (late genes) which are repressed by the ecdysone receptor. The early class of genes are thought to have two functions reciprocal to those of the ecdysone receptor; the repression of the early transcripts and the induction of late gene transcription. Members of the early genes so far isolated and characterised belong to the class of molecules with characteristics similar to known transcription factors. They are thus predicted to behave as expected by the model of ecdysone action (Ashburner, 1991). More recently, the early genes E74 and E75 have been shown to bind both types of ecdysone inducible genes (Thummel et al., 1990; Segraves and Hogness, 1991), thus supporting their proposed dual activities. It should be noted however, that the activation of a hierarchy of genes is not limited to third instar larvae salivary glands, but that the response to the ecdysone peak at the end of larval life is observed in many other tissues, such as the imaginal disks (i.e. those tissues which metamorphose to adult structures) and other larval tissues which histolyse at the end of larval life (eg. larval fat body). The model for ecdysone action as deduced by studying the third instar chromosome puffing may not apply to the activation of ecdysone regulated genes in adults. In other words, the requirement for other factors in addition to the active ecdysone receptor must be satisfied for correct developmental expression (e.g. the Drosophila yolk protein gene expression in adults is under control of doublesex, the last gene in the sex determination gene hierarchy).
The ecdysone receptor and the early gene E75 belong to the steroid receptor superfamily. Other Drosophila genes, including ultraspiracle, tailless, sevenup and FTZ-FI, also belong to this family. However, of all these genes only the ecdysone receptor is known to have a ligand, and thus the others are known as orphan receptors. Interestingly, despite the ultraspiracle protein ligand binding region sharing 49% identity with the vertebrate retinoic X receptor (RXR) ligand binding region (Oro et al., 1990), they do not share the same ligand (i.e. the RXR ligand is 9-cis retinoic acid) (Heymann et al., 1992 and Mangelsdorf et al., 1992). All the Drosophila genes mentioned are involved in development, ultraspiracle for example, is required for embryonic and larval abdominal development. The protein products of these genes all fit the main features of the steroid receptor superfamily (Evans, 1988; Green and Chambon, 1988, Beato, 1989) i.e. they have a variable N terminus region involved in ligand independent transactivation (Domains A and B), a highly conserved 66-68 amino acid region which is responsible for the binding of DNA at specific sites (Domain C), a hinge region thought to contain a nuclear translocation signal (Domain D), and a well conserved region containing the ligand binding region, transactivation sequences and the dimerisation phase (Domain E). The last region, domain F, is also very variable and its function is unknown.
Steroid receptor action has been elucidated in considerable detail in vertebrate systems at both the cellular and molecular levels. In the absence of ligand, the receptor molecule resides in the cytoplasm where it is bound by Hsp90, Hsp70 and p59 to form the inactive complex (Evans, 1988). Upon binding of the ligand molecule by the receptor a conformational change takes place which releases the Hsp90, Hsp70 and p59 molecules, while exposing the nuclear translocation signals in the receptor. The ligand dependent conformational change is seen in the ligand binding domain of both progesterone and retinoic acid receptors (Allan et al., 1992a). This conformational change has been further characterised in the progesterone receptor and was found to be indispensable for gene transactivation (Allan et al., 1992b). Once inside the nucleus the receptor dimer binds to the receptor responsive element at a specific site on the DNA resulting in the activation or repression of a target gene. The receptor responsive elements usually consist of degenerate direct repeats, with a spacer between 1 and 5 nucleotides, which are bound by a receptor dimer through the DNA binding region (Domain C).
Whereas some steroid hormone receptors are active as homodimers others act as heterodimers. For example, in vertebrates, the retinoic acid receptor (RAR) forms heterodimers with the retinoic X receptor (RXR). RXR can also form heterodimers with the thyroid receptor, vitamin D receptor (Yu et al., 1991; Leid et al., 1992) and peroxisome activator receptor (Kliewer et al., 1992). Functionally the main difference between homodimers and heterodimers is increased specificity of binding to specific response elements. This indicates that different pathways can be linked, co-ordinated and modulated, and more importantly this observation begins to explain the molecular basis of the pleotropic activity of retinoic acid in vertebrate development (Leid et al., 1992b). Similarly, the Drosophila ultraspiracle gene product was recently shown to be capable of forming heterodimers with retinoic acid, thyroid, vitamin D and peroxisome activator receptors and to stimulate the binding of these receptors to their target responsive elements (Yao et al., 1993). More significantly, the ultraspiracle gene product has also been shown to form heterodimers with the ecdysone receptor, resulting in cooperative binding to the ecdysone response element and capable of rendering mammalian cells ecdysone responsive (Yao et al., 1992). The latter is of importance since transactivation of the ecdysone gene alone in mammalian cells fails to elicit an ecdysone response (Koelle et al., 1991), therefore suggesting that the ultraspiracle gene product is an integral component of a functional ecdysone receptor (Yao et al., 1992). It is possible that the ultraspiracle product competes with other steroid receptors or factors to form heterodimers with the ecdysone receptor. Moreover it remains to be investigated if ultraspiracle is expressed in all tissues of the Drosophila larvae. Despite ultraspiracle being necessary to produce a functional ecdysone receptor, the mechanism by which this activation takes place is as yet undetermined.
We have now isolated and characterised the ecdysone steroid receptor from Heliothis virescens (hereinafter HEcR). We have found that surprisingly unlike the Drosophila ecdysone steroid receptor (hereinafter DEcR), in reports to-date, HEcR can be induced by known non-steroidal inducers. It will be appreciated that this provides many advantages for the system.
Steroids are difficult and expensive to make. In addition, the use of a non-steroid as the inducer allows the system to be used in agrochemical and pharmaceutical applications, not least because it avoids application of a steroid which is already present in insects and/or mammals. For example, it would not be feasible to use a gene switch in a mammalian cell which was induced by a naturally occurring steroidal inducer. It will also be appreciated that for environmental reasons it is advantageous to avoid the use of steroids as inducers.
According to one aspect of the present invention there is provided DNA having the sequence shown in SEQ ID NO: 2, wherein SEQ ID NO: 2 gives the sequence for the HEcR.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 2, which encodes for the HEcR ligand binding domain.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 2, which encodes for the HEcR DNA binding domain.
According to yet another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 2, which encodes for the HEcR transactivation domain.
According to a further aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 2, which encodes for the HEcR hinge domain.
According to a still further aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 2, which encodes for the HEcR carboxy terminal region.
According to one aspect of the present invention there is provided DNA having the sequence shown in SEQ ID NO: 3, wherein SEQ ID NO: 3 gives the sequence for the HEcR.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 3, which encodes for the HEcR ligand binding domain.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 3, which encodes for the HEcR DNA binding domain.
According to yet another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 3, which encodes for the HEcR transactivation domain.
According to a further aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 3, which encodes for the HEcR hinge domain.
According to a still further aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 3, which encodes for the HEcR carboxy terminal region.
According to one aspect of the present invention there is provided DNA having the sequence shown in SEQ ID NO: 4, wherein SEQ ID NO: 4 gives the sequence for the HEcR.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 4, which encodes for the HEcR ligand binding domain.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 4, which encodes for the HEcR DNA binding domain.
According to yet another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 4, which encodes for the HEcR transactivation domain.
According to a further aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 4, which encodes for the HEcR hinge domain.
According to a still further aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 4, which encodes for the HEcR carboxy terminal region.
As mentioned above, steroid receptors are eukaryotic transcriptional regulatory factors which, in response to the binding of the steroid hormone, are believed to bind to specific DNA elements and activate transcription. The steroid receptor can be divided into six regions, designated A to F, using alignment techniques based on shared homology with other members of the steroid hormone receptor superfamily. Krust et al identified two main regions in the receptor, C and E. Region C is hydrophilic and is unusual in its high content in cysteine, lysine and arginine.It corresponds to a DNA-binding domain, sometimes referred to as the xe2x80x9czinc fingerxe2x80x9d. It is the DNA binding domain which binds to the upstream DNA of the responsive gene. Such upstream DNA is known as the hormone response element or HRE for short. Region E is hydrophobic and is identified as the hormone (or ligand) binding domain. Region E can be further subdivided into regions E1, E2 and E3.
The region D, which separates domains C and E is highly hydrophobic and is flexible. It is believe that communication between domains E and C involves direct contact between them through region D, which provides a hinge between the two domains. Region D is therefore referred to as the hinge domain.
The mechanism of the receptor appears to require it to interact with some element(s) of the transcription machinery over and above its interactions with the hormone and the hormone response element. N-terminal regions A and B perform such a function and are jointly known as the transactivation domain. The carboxy terminal region is designated F.
The domain boundaries of the HEcR can be defined as follows:
The DNA binding domain is very well defined and is 66 amino acids long, thus providing good boundaries. The above intervals have been defined using the multiple alignment for the ecdysone receptors (FIG. 5).
The present invention also includes DNA which shows homology to the sequences of the present invention. Typically homology is shown when 60% or more of the nucletides are common, more typically 65%, preferably 70%, more preferably 75%, even more preferably 80% or 85%, especially preferred are 90%, 95%, 98% or 99% or more homology.
The present invention also includes DNA which hybridises to the DNA of the present invention and which codes for at least part of the Heliothis ecdysone receptor transactivation domain, DNA binding domain, hinge domain, ligand binding domain and/or carboxy terminal region. Preferably such hybridisation occurs at, or between, low and high stringency conditions. In general terms, low stringency conditions can be defined as 3xc3x97SCC at about ambient temperature to about 65xc2x0 C., and high stringency conditions as 0.1xc3x97SSC at about 65xc2x0 C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3xc3x97SSC is three time as strong as SSC and so on.
The present invention further includes DNA which is degenerate as a result of the genetic code to the DNA of the present invention and which codes for a polypeptide which is at least part of the Heliothis ecdysone receptor transactivation domain, DNA binding domain, hinge domain, ligand binding domain and/or carboxy terminal region.
The DNA of the present invention may be cDNA or DNA which is in an isolated form.
According to another aspect of the present invention there is provided a polypeptide comprising the Heliothis ecdysone receptor or a fragment thereof, wherein said polypeptide is substantially free from other proteins with which it is ordinarily associated, and which is coded for by any of the DNA of the present invention.
According to another aspect of the present invention there is provided a polypeptide which has the amino acid sequence of SEQ ID NO: 4 or any allelic variant or derivative thereof, wherein SEQ ID No. 4 gives the amino acid sequence of the HEcR polypeptide.
According to another aspect of the present invention there is provided a polypeptide which has part of the amino acid sequence of SEQ ID No. 4 or any allelic variant or derivative thereof, which sequence provides the HEcR ligand binding domain.
According to another aspect of the present invention there is provided a polypeptide which has part of the amino acid sequence of SEQ ID No. 4 or any allelic variant or derivative thereof, which sequence provides the HEcR DNA binding domain.
According to yet another aspect of the present invention there is provided a polypeptide which has part of the amino acid sequence of SEQ ID No. 4 or any allelic variant or derivative thereof, which sequence provides the HEcR transactivation domain.
According to a further aspect of the present invention there is provided a polypeptide which has the amino acid sequence of a part of SEQ ID No. 4 or any allelic variant or derivative thereof, which sequence provides the HEcR hinge domain.
According to a still further aspect of the present invention there is provided a polypeptide which has the amino acid sequence of a part of SEQ ID No. 4 or any allelic variant or derivative thereof, which sequence provides the HEcR carboxy terminal region.
For the avoidance of doubt, spliced variants of the amino acid sequences of the present invention are included in the present invention.
Preferably, said derivative is a homologous variant which has conservative amino acid changes. By conservation amino acid changes we mean replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An examples of such a change is the replacement of valine by methionine and vice versa.
According to another aspect of the present invention there is provided a fusion polypeptide comprising at least one of the polypeptides of the present invention functionally linked to an appropriate non-Heliothis ecdysone receptor domain(s).
According to an especially preferred embodiment of the present invention the HEcR ligand binding domain of the present invention is fused to a DNA binding domain and a transactivation domain.
According to another embodiment of the present invention the DNA binding domain is fused to a ligand binding domain and a transactivation domain.
According to yet another embodiment of the present invention the transactivation domain is fused to a ligand binding domain and a DNA binding domain.
The present invention also provides recombinant DNA encoding for these fused polypeptides.
According to an especially preferred embodiment of the present invention there is provided recombinant nucleic acid comprising a DNA sequence encoding the HEcR ligand binding domain functionally linked to DNA encoding the DNA binding domain and transactivation domain from a glucocorticoid receptor.
According to yet another aspect of the present invention there is provided recombinant nucleic acid comprising a DNA sequence comprising a reporter gene operably linked to a promoter sequence and a hormone response element which hormone response element is responsive to the DNA bonding domain encoded by the DNA of of the present invention.
According to another aspect of the present invention there is provided a construct transformed with nucleic acid, recombinant DNA, a polypeptide or a fusion polypeptide of the present invention. Such constructs include plasmids and phages suitable for transforming a cell of interest. Such constructs will be well known to those skilled in the art.
According to another aspect of the present invention there is provided a cell transformed with nucleic acid, recombinant DNA, a polypeptide, or a fusion polypeptide of the present invention.
Preferably the cell is a plant, fungus or mammalian cell.
For the avoidance of doubt fungus includes yeast.
The present invention therefore provides a gene switch which is operably linked to a foreign gene or a series of foreign genes whereby expression of said foreign gene or said series of foreign genes may be controlled by application of an effective exogenous inducer.
Analogs of ecdysone, such as Muristerone A, are found in plants and disrupt the development of insects. It is therefore proposed that the receptor of the present invention can be used be in plants transformed therewith as an insect control mechanism. The production of the insect-damaging product being controlled by an exogenous inducer. The insect-damagin g product can be ecdysone or another suitable protein.
The first non-steroidal ecdysteroid agonists, dibenzoyl hydrazines, typified by RH-5849 [1,2-dibenzoyl, 1-tert-butyl hydrazide], which is commercially available as an insecticide from Rohm and Haas, were described back in 1988. Another commercially available compound in this series is RH-5992 [tebufenozide, 3,5-dimethylbenzoic acid 1-1 (1,1-dimethylethyl)-2(4-ethylbenzoyl) hydrazide]. These compounds mimic 20-hydroxyecdysone (20E) in both Manduca sexta and Drosophila melanogaster. These compounds have the advantage that they have the potential to control insects using ecdysteroid agonists which are non-steroidal. Further Examples of such dibenzoyl hydrazines are given in U.S. Pat. No. 5,117,057 to Rohm and Haas, and Oikawa et al, Pestic Sci, 41, 139-148 (1994). However, it will be appreciated that any inducer of the gene switch of the present invention, whether steroidal or non-steroidal, and which is currently or becomes available, may be used.
The gene switch of the present invention, then, when linked to an exogenous or foreign gene and introduced into a plant by transformation, provides a means for the external regulation of expression of that foreign gene. The method employed for transformation of the plant cells is not especially germane to this invention and any method suitable for the target plant may be employed. Transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature such as agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection or plants cells and protoplasts, microprojectile transformation, to mention but a few. Reference may be made to the literature for full details of the known methods.
Neither is the plant species into which the chemically inducible sequence is inserted particularly germane to the invention. Dicotyledonous and monocotyledonous plants can be transformed. This invention may be applied to any plant for which transformation techniques are, or become, available. The present invention can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, and cotton; cereals such as wheat, barley, rice, maize, and sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons; and vegetables such as carrot, lettuce, cabbage and onion. The switch is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues.
In a particularly preferred embodiment of the present invention, the gene switch of the present invention is used to control expression of genes which confer resistance herbicide resistance and/or insect tolerance to plants.
Recent advances in plant biotechnology have resulted in the generation of transgenic plants resistant to herbicide application, and transgenic plants resistant to insects. Herbicide tolerance has been achieved using a range of different transgenic strategies. One well documented example in the herbicide field is the use the bacterial xenobiotic detoxifying gene phosphinothricin acetyl transferase (PAT) from Streptomyces hydroscopicus. Mutated genes of plant origin, for example the altered target site gene encoding acetolactate synthase (ALS) from Arabidopsis, have been successfully utilised to generate transgenic plants resistant to herbicide application. The PAT and ALS genes have been expressed under the control of strong constitutive promoter. In the field of insecticides, the most common example to-date is the use of the Bt gene.
We propose a system where genes conferring herbicide and/or insect tolerance would be expressed in an inducible manner dependent upon application of a specific activating chemical. This approach has a number of benefits for the farmer, including the following:
1. Inducible control of herbicide and/or insect tolerance would alleviate any risk of yield penalties associated with high levels of constitutive expression of herbicide and/or insect resistance genes. This may be a particular problem as early stages of growth where high levels of transgene product may directly interfere with normal development. Alternatively high levels of expression of herbicide and/or insect resistance genes may cause a metabolic drain for plant resources.
2. The expression of herbicide resistance genes in an inducible manner allows the herbicide in question to be used to control volunteers if the activating chemical is omitted during treatment.
3. The use of an inducible promoter to drive herbicide and/or insect resistance genes will reduce the risk of resistance becoming a major problem. If resistance genes were passed onto weed species from related crops, control could still be achieved with the herbicide in the absence of inducing chemical. This would particularly be relevant if the tolerance gene confirmed resistance to a total vegetative control herbicide which would be used (with no inducing chemical) prior to sowing the crop and potentially after the crop has been harvested. For example, it can be envisaged that herbicide resistance cereals, such as wheat, might outcross into the weed wild oats, thus conferring herbicide resistance to this already troublesome weed. A further example is that the inducible expression of herbicide resistance in sugar beet will reduce the risk of wild sugar beet becoming a problem. Similarly, in the field of insect control, insect resistance may well become a problem if the tolerance gene is constitutively expressed. The used of an inducible promoter will allow a greater range of insect resistance control mechanisms to be employed.
This strategy of inducible expression of herbicide resistance can be achieved with a pre-spray of chemical activator or in the case of slow acting herbicides, for example N-phosphonomethyl-glycine (commonly known as glyphosate), the chemical inducer can be added as a tank mix simultaneously with the herbicide. Similar strategies can be employed for insect control.
This strategy can be adopted for any resistance confering gene/corresponding herbicide combination, which is, or becomes, available. For example , the gene switch of the present invention can be used with:
1. Maize glutathione S-transferase (GST-27) gene (see our International Patent Publication No WO90/08826), which confers resistance to chloroacetanilide herbicides such as acetochlor, metolachlor and alachlor.
2. Phosphinotricin acetyl transferase (PAT), which confers resistance to the herbicide commonly known as glufosinate.
3. Acetolactate synthase gene mutants from maize (see our International Patent Publication No WO90/14000) and other genes, which confer resistance to sulphonyl urea and imadazolinones.
4. Genes which confer resistance to glyphosate. Such genes include the glyphosate oxidoreductase gene (GOX) (see International Patent Publication No. WO92/00377); genes which encode for 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSPS), including Class I and Class II EPSPS, genes which encode for mutant EPSPS, and genes which encode for EPSPS fusion peptides such as that comprised of a chloroplast transit peptide and EPSPS (see for example EP 218 571, EP 293 358, WO91/04323, WO92/04449 and WO92106201); and genes which are involved in the expression of CPLyase.
Similarly, the strategy of inducible expression of insect resistance can be adopted for any tolerance confering gene which is, or becomes, available.
The gene switch of the present invention can also be used to controlled expression of foreign proteins in yeast and mammalian cells. Many heterologous proteins for many applications are produced by expression in genetically engineered bacteria, yeast cells and other eucaryotic cells such as mammalian cells.
As well as the obvious advantage in providing control over the expression of foreign genes in such cells, the switch of the present invention provides a further advantage in yeasts and mammalian cells where accumulation of large quantities of an heterologous protein can damage the cells, or where the heterologous protein is damaging such that expression for short periods of time is required in order to maintain the viability of the cells.
Such an inducible system also has applicability in gene therapy allowing the timing of expression of the therapeutic gene to be controlled. The present invention is therefore not only applicable to transformed mammalian cells but also to mammals per se.
A further advantage of the inducible system of the present invention in mammalian cells is that, because it is derived from a insect, there is less chance of it being effected by inducers which effect the natural mammalian steroid receptors.
In another aspect of the present invention the gene switch is used to switch on genes which produce potentially damaging or lethal proteins. Such a system can be employed in the treatment of cancer in which cells are transformed with genes which express proteins which are lethal to the cancer. The timing of the action of such proteins on the cancer cells can be controlled using the switch of the present invention.
The gene switch of the present invention can also be used to switch genes off as well as on. This is useful in disease models. In such a model the cell is allowed to grow before a specific gene(s) is switched off using the present invention. Such a model facilitates the study of the effect of a specific gene(s).
Again the method for producing such transgenic cells is not particularly germane to the present invention and any method suitable for the target cell may be used; such methods are known in the art, including cell specific transformation.
As previously mentioned, modulation of gene expression in the system appears in response to the binding of the HEcR to a specific control, or regulatory, DNA element. A schematic representation of the HEcR gene switch is shown in FIG. 6. For ease of reference, the schematic representation only shows three main domains of the HEcR, namely the transactivation domain, DNA binding domain and the ligand binding domain. Binding of a ligand to the ligand binding domain enables the DNA binding domain to bind to the HRE resulting in expression (or indeed repression) of a target gene.
The gene switch of the present invention can therefore be seen as having two components. The first component comprising the HEcR and a second component comprising an appropriate HRE and the target gene. In practice, the switch may conveniently take the form of one or two sequences of DNA. At least part of the one sequence, or one sequence of the pair, encoding the HEcR protein. Alternatively, the nucleic acid encoding the HEcR can be replaced by the protein/polypeptide itself.
Not only does the switch of the present invention have two components, but also one or more of the domains of the receptor can be varied producing a chimeric gene switch. The switch of the present invention is very flexible and different combinations can be used in order to vary the result/to optimise the system. The only requirement in such chimeric systems is that the DNA binding domain should bind to the hormone response element in order to produce the desired effect.
The glucocorticoid steroid receptor is well characterised and has been found to work well in plants. A further advantage of this receptor is that it functions as a homodimer. This means that there is no need to express a second protein such as the ultraspiracle in order to produce a functional receptor. The problem with the glucocorticoid steroid receptor is that ligands used to activate it are not compatible with agronomic practice.
In a preferred aspect of the present invention the receptor comprises glucocorticoid receptor DNA binding and transactivation domains with a Heliothis ligand binding domain according to the present invention. The response unit preferably comprising the glucocorticoid hormone response element and the desired effect gene. In the Examples, for convenience, this effect gene took the form of a reporter gene. However, in non-test or non-screen situations the gene will be the gene which produces the desired effect, for example produces the desired protein. This protein may be a natural or exogenous protein. It will be appreciated that this chimeric switch combines the best features of the glucocorticoid system, whilst overcoming the disadvantage of only being inducible by a steroid.
In another preferred embodiment, the Heliothis ligand binding domain is changed, and preferably replaced with a non-Heliothis ecdysone receptor ligand binding domain. For example, we have isolated suitable sequences from Spodoptera exigua. 
Thus, according to another aspect of the present invention there is provided DNA having the sequence shown in SEQ ID NO: 6.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 6, which encodes for the Spodoptera ecdysone ligand binding domain.
According to another aspect of the present invention there is provided DNA having part of the sequence shown in SEQ ID NO: 6, which encodes for the Spodoptera ecdysone hinge domain.
The present invention also provides the polypeptides coded for by the above DNA sequences of SEQ ID NO: 6.
A further advantage with such chimeric systems is that they allow you to choose the promoter which is used to drive the effector gene according to the desired end result. For example, placing the foreign gene under the control of a cell specific promoter can be particularly advantageous in circumstances where you wish to control not only the timing of expression, but also which cells expression occurs in. Such a double control can be particularly important in the areas of gene therapy and the use of cytotoxic proteins.
Changing the promoter also enables gene expression to be up- or down-regulated as desired.
Any convenient promoter can be used in the present invention, and many are known in the art.
Any convenient transactivation domain may also be used. The transactivation domain VP16 is a strong activator from Genentech Inc., and is commonly used when expressing glucocorticoid receptor in plants. Other transactivation domains derived for example from plants or yeast may be employed.
In a preferred embodiment of the present invention, the DNA binding domain is the glucocorticoid DNA binding domain. This domain is commonly a human glucocorticoid receptor DNA binding domain. However, the domain can be obtained from any other convenient source, for example, rats.
According to another aspect of the present invention there is provided a method of selecting compounds capable of being bound to an insect steroid receptor superfamily member comprising screening compounds for binding to a polypeptide or fusion polypeptide of the present invention, and selecting said compounds exhibiting said binding.
According to another aspect of the present invention there is provided a compound selected using the method of the present invention.
According to another aspect of the present invention there is provided an agricultural or pharmaceutical composition comprising the compound of the present invention.
According to yet another aspect of the present invention there is provided the use of the compound of the present invention as a pesticide, pharmaceutical and/or inducer of the switch. It will be appreciated that such inducers may well be useful as insecticides in themselves.
According to a further aspect of the present invention there is provided a method of producing a protein or peptide or polypeptide comprising introducing into a cell of the present invention, a compound which binds to the ligand binding domain in said cell.
Various preferred features and embodiments of the present invention will now be described by way of non-limiting example with reference to the accompanying examples and figures, in which figures: